Non-Coding RNA最新文献

{"title":"Long Noncoding RNA <i>H19</i> Inhibits Differentiation and Function of Secretory Lineage Cells in the Intestinal Epithelium by Targeting ATOH1.","authors":"Min S Kwon, Hee K Chung, Hongxia Chen, Amy VanderStoep, Bridgette Warner, Haonan Zhao, Prathik Atluri, Emnet Tesfayohannes, Douglas J Turner, Lan Xiao, Jian-Ying Wang","doi":"10.3390/ncrna12050037","DOIUrl":"10.3390/ncrna12050037","url":null,"abstract":"<p><p><b>Background/Objectives</b>: Intestinal mucosal function relies on the integrated contribution of multiple cell lineages, including secretory cells. Long noncoding RNA <i>H19</i> modulates many cell processes essential for human pathologies. Here, we investigated the role of <i>H19</i> in development and function of secretory lineage cells and elucidated the implication of altered ATOH1 by <i>H19</i> in dysfunction of the secretory cells. <b>Methods</b>: Studies were conducted in <i>H19</i>-transgenic (H19-Tg) and <i>H19</i>-knockout (H19^-/-) mice, intestinal organoids, and Caco-2 cells. Secretory lineage cells were examined by fluorescent immunostaining with their specific cell markers, including Lysozyme, Mucin2, DCLK1, and Chromogranin A. Intestinal mucosal growth was measured by BrdU incorporation, and intestinal permeability was detected by paracellular tracer flux assay using FITC-Dextran. Single cell RNA-sequence, quantitative real-time PCR, and Western immunoblotting analyses were used to monitor changes in gene expression. <b>Results</b>: Tissue-specific transgenic expression of <i>H19</i> in the intestinal epithelium caused defects in Paneth, goblet, tuft, and enteroendocrine cells in mice, whereas targeted deletion of the <i>H19</i> gene increased the function of secretory lineage cells. Locally increasing the levels of <i>H19</i> in the epithelium impaired gut barrier function in H19-Tg mice, although it failed to alter mucosal growth. The intestinal mucosal tissues of H19-Tg mice exhibited a remarkable decrease in the levels of differentiation-associated transcription factors including ATOH1. Ectopically expressed ATOH1 in the <i>H19</i>-enriched intestinal organoids derived from H19-Tg mice rescued development of the secretory lineage cells. Mechanistically, <i>H19</i> inhibited ATOH1 expression via inhibition of its translation. <b>Conclusions</b>: These findings indicate that <i>H19</i> functions as a negative regulator of differentiation and function of secretory lineage cells in the intestinal mucosa at least partially via the inhibition of ATOH1 translation.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 5","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600228/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149031107","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Transcriptome-Wide Analysis Nominates DIO3OS and C9orf139 as Survival-Associated Long Non-Coding RNAs in Glioblastoma.","authors":"Minseon Kim, Seung-Kyoon Kim, Jaeil Han","doi":"10.3390/ncrna12050036","DOIUrl":"10.3390/ncrna12050036","url":null,"abstract":"<p><p><b>Background</b>: Long non-coding RNAs (lncRNAs) are widely proposed as determinants of temozolomide response in glioblastoma (GBM), but many nominations rest on analyst-derived survival endpoints, screens unadjusted for O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, and models evaluated with the same data used to select them. We asked which lncRNA-outcome associations remain when endpoint definition, confounder adjustment, and external validation are addressed explicitly. <b>Methods</b>: We analyzed 94 IDH-wildtype, temozolomide-treated primary GBM patients from The Cancer Genome Atlas (TCGA), using the rule-based progression-free interval (PFI) of the TCGA Pan-Cancer Clinical Data Resource and overall survival (OS) as separate endpoints. A joint transcriptome-wide discovery for OS was performed across TCGA and the identically processed CPTAC-3 cohort (n = 282, 209 deaths), the latter unselected for IDH status because IDH annotation is unavailable for CPTAC-3, with adjustment for MGMT promoter methylation status, cellular composition, and expression subtype. Nominated transcripts were tested for external validation in two independent CGGA cohorts, and incremental discrimination was assessed as the optimism-corrected change in C-index (ΔC-index), benchmarked against the gain expected from a transcriptome-wide search alone. <b>Results</b>: No lncRNA was associated with PFI at a transcriptome-wide false-discovery rate (FDR) below 0.05. The joint OS discovery nominated DIO3OS, the only transcript reaching transcriptome-wide significance (hazard ratio 1.38 per standard deviation, 95% confidence interval 1.20-1.58, FDR 0.022), and C9orf139 (FDR 0.065); both were independently significant in each discovery cohort and robust to adjustment for MGMT status, cellular composition, and expression subtype. Both, however, were null with reversed point estimates in the two CGGA cohorts, were unassociated with PFI, and added only modest optimism-corrected discrimination (ΔC-index 0.07-0.11), close to what a transcriptome-wide search alone generates. Replication was thus confined to two cohorts sharing a processing pipeline. <b>Conclusions</b>: We nominate DIO3OS and C9orf139 as candidate survival-associated lncRNAs in GBM, requiring prospective evaluation in cohorts with complete IDH annotation and progression endpoints, and show how endpoint definition, selection optimism, and external validation determine which lncRNA associations survive scrutiny.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 5","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600001/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149031123","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"microRNAs as Regulators of the Immune Response and Their Potential Therapeutic Applications in Cancer.","authors":"Ezgi Biltekin, Bulent Ozpolat","doi":"10.3390/ncrna12050034","DOIUrl":"10.3390/ncrna12050034","url":null,"abstract":"<p><p>MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as critical regulators of gene expression and key cellular processes in cancer, including tumor cell proliferation, survival, metastasis, and both innate and adaptive immune responses within the tumor microenvironment. Through their ability to modulate immune cell development, differentiation, polarization, antigen presentation, cytokine signaling and immune checkpoint expression, miRNAs play a vital role in shaping immune responses and represent attractive therapeutic candidates for remodeling the tumor immune microenvironment. This review provides a comprehensive overview of the mechanisms through which miRNAs regulate anti-tumor immunity and contribute to immune evasion with particular emphasis on their role in modulating major immune checkpoint pathways, including PD-1/PD-L1, CTLA-4, LAG-3, TIM-3 and CD28-mediated signaling. We further examine the contribution of miRNAs to therapeutic resistance and discuss their potential integration with immune checkpoint blockade, chemotherapy, PARP inhibitors and targeted therapies to enhance antitumor efficacy and overcome treatment resistance. Recent advances in spatial transcriptomics and computational approaches have expanded our understanding of miRNA-mediated regulatory networks at single-cell and tissue levels and provide new insights into tumor-immune interactions. In addition, we evaluate the current progress in the clinical development of miRNA-based therapeutics, including miRNA mimics and inhibitors and discuss the major challenges associated with efficient delivery, off-target effects, immunogenicity and patient heterogeneity. Collectively, the evidence highlights the growing potential of miRNAs as diagnostic biomarkers, therapeutic targets and therapeutic tools in cancer immunotherapy. Continued advances in RNA therapeutics, delivery technologies and multi-omics approaches are expected to accelerate the clinical translation of miRNA-based strategies for personalized cancer therapy.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 5","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600176/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149031130","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cerebrospinal Fluid miRNA Profiling as a Potential Liquid Biopsy for Vestibular Schwannomas.","authors":"Małgorzata Litwiniuk-Kosmala, Maria Makuszewska, Robert Bartoszewicz, Agnieszka Jasińska-Nowacka, Maciej Ołdak, Bartłomiej Gielniewski, Bartosz Wojtaś, Kazimierz Niemczyk","doi":"10.3390/ncrna12050035","DOIUrl":"10.3390/ncrna12050035","url":null,"abstract":"<p><p><b>Background/Objectives</b>: This study aimed to identify a characteristic miRNA expression profile in the CSF of patients diagnosed with vestibular schwannoma and evaluate its potential for tumor assessment. <b>Methods</b>: In this prospective study, 17 CSF and corresponding tumor samples (seven small tumors-SVS and 10 large tumors-LVS) were collected from patients operated on for VS in a Tertiary Academic Center. The miRNA expression was analyzed using high-throughput RNA sequencing (NovaSeq 6000 Illumina). Data were normalized, and a comparative analysis of miRNA expression rankings was performed between VS patients and a public healthy donor dataset. Functional implications were explored using KEGG pathway enrichment analysis. <b>Results</b>: A total of 1633 miRNAs were identified in all CSF samples derived from VS patients. Comparison with healthy donors revealed a moderate ranking correlation (ρ = 0.39), with significant shifts for specific molecules like hsa-miR-766-3p and hsa-miR-182-5p. Only six miRNAs were found to correlate between CSF and tumor tissue, while 16 exhibited a negative correlation. No statistical correlation was found between tumor size and the CSF miRNA profile. KEGG analysis highlighted enriched pathways, including neurotrophin signaling and focal adhesion. <b>Conclusions</b>: The results of our study support the feasibility of miRNA-based CSF liquid biopsy for VS assessment. However, the results of miRNA expression profiling conducted in tumor tissue cannot be directly transferred into CSF sample analyses. Further studies are warranted to explain this phenomenon and to search for reliable miRNA markers of VS progression in the CSF liquid biopsy specimens.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 5","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600145/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149031118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Circulating Cell-Free miR-25-3p Is Upregulated in Brazilian Patients with Prostate Cancer and Contributes to Tumor Aggressiveness.","authors":"Monyse Nobrega, Hellen Kuasne, Marilesia Ferreira de Souza, Mariana Bisarro Dos Reis, Larissa Cristina Bastos de Oliveira, Paulo Emilio Fuganti, Morag Park, Ilce Mara de Syllos Cólus","doi":"10.3390/ncrna12050033","DOIUrl":"10.3390/ncrna12050033","url":null,"abstract":"<p><p><b>Background/Objectives:</b> Prostate cancer (PCa) has a high incidence worldwide, yet reliable prognostic biomarkers to distinguish indolent from aggressive forms are lacking. MicroRNAs (miRNAs), a class of small non-coding RNAs, are stable molecules that play an important role in cancer development and can be detected in circulation by non-invasive methods. This study aimed to examine the influence of the cell-free (cf) miRNAs levels on PCa risk and aggressiveness using plasma samples and cell models. <b>Methods:</b> Four overexpressed miRNAs in prostate cancer tissue were selected from The Cancer Genome Atlas database: miR-25-3p, miR-92a-1-5p, miR-92a-2-5p, and miR-148a-3p. Cf-miRNA levels were validated via quantitative reverse transcription polymerase chain reaction (RT-qPCR) in plasma samples from 80 PCa patients and 40 controls in a Brazilian cohort. Clinicopathological associations were analyzed to determine prognostic value. Subsequently, functional analyses were performed using transient transfection of miR-25-3p in the LNCaP cell line, assessing proliferation, migration, invasion, and target gene regulation. <b>Results:</b> Among the miRNAs analyzed, only miR-25-3p was upregulated in the plasma of PCa patients compared to controls (<i>p</i> = 0.013). Patients with International Society of Urological Pathology (ISUP) grade ≥2 presented higher expression levels of miR-25-3p (<i>p</i> = 0.020) compared to patients with ISUP grade 1. Overexpression of miR-25-3p significantly increased cell proliferation, colony formation, migration, and invasion capacity and modulated the expression of <i>BCL2L11</i>, <i>CDH1</i>, <i>CDKN1C</i>, <i>EHZ2</i>, and <i>TP53</i> genes. <b>Conclusions:</b> miR-25-3p had an oncogenic role in PCa, showing a potential marker to assess PCa development and progression using liquid biopsy.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 5","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600208/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149031060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exosomal lncRNAs in Cerebrovascular Diseases: Biomarkers, Pathological Mechanisms, and Therapeutic Potential.","authors":"Haiyu Su, Daiju Tao, Jia Teng, Li Zhang, Ying Shen, Renhua Yang, Jiarui Yang, Rongji Sun, Zhiqiang Shen, Peng Chen, Bo He","doi":"10.3390/ncrna12050032","DOIUrl":"10.3390/ncrna12050032","url":null,"abstract":"<p><p><b>Background:</b> Cerebrovascular diseases have complex pathogenesis and pose a serious threat to human health; thus, novel diagnostic and therapeutic strategies are needed. Small extracellular vesicles (sEVs), commonly referred to as exosomes, are 30-150 nm lipid-bilayer vesicles that shield long noncoding RNAs (lncRNAs) from degradation. Because exosomal lncRNAs are more stable than free lncRNAs in blood and cerebrospinal fluid and can cross the blood-brain barrier, they are promising as biomarkers and therapeutic vectors. This review summarizes the roles and mechanisms of exosomal lncRNAs in cerebrovascular diseases. <b>Methods:</b> This narrative review is based on the experimental literature and focuses on the biological functions and regulatory mechanisms of exosomal lncRNAs in cerebrovascular disorders. <b>Results:</b> As competing endogenous RNAs (ceRNAs), they sequester microRNAs (miRNAs), thereby derepressing downstream target-gene expression and modulating neuronal injury (oxidative stress, apoptosis, neuroinflammation) through the nuclear factor kappa-B (NF-κB) and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathways. They show altered profiles in acute stroke (ischemic/hemorrhagic) correlated with neurological deficits, and are relevant to the early diagnosis of chronic diseases (atherosclerosis, aneurysm) and vascular dementia. <b>Conclusions:</b> Exosomal lncRNAs demonstrate promising translational potential in preclinical studies because they combine exosome delivery capabilities with lncRNA regulatory functions, although clinical validation remains limited.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 5","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13600189/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149031070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Long-Read RNA Sequencing Reveals an Isoform Switching Pattern in Healthy Microglial Cell Lines Exposed to Radiotherapy.","authors":"Tatiana Zuppelli, Laura Orlando, Giuseppe N Conti, Sofia P Lombardo, Lucia Longhitano, Giulia Chisari, Cesarina Giallongo, Francesco Bellia, Maria Gabriella Sabini, Stefania Mele, Ignazio A Barbagallo, Nunzio Vicario, Rosalba Parenti, Michelino Di Rosa, Enrico La Spina, Stefano Forte, Daniele Tibullo, Giovanni Li Volti","doi":"10.3390/ncrna12040031","DOIUrl":"10.3390/ncrna12040031","url":null,"abstract":"<p><p><b>Background/Objectives</b>: Radiotherapy (RT) is a fundamental treatment for brain tumors but often leads to long-term neurotoxicity, partly caused by microglial dysfunction. In this study, we used long-read RNA sequencing (LR-RNA-seq) to map the isoform-specific transcriptome of human microglial HMC3 cells after RT exposure. <b>Methods</b>: The HMC3 human microglial cell line was treated with standard radiotherapy and, after RNA extraction and library preparation, LR-RNA-seq was performed. Further bioinformatic analyses allowed to investigate on the isoform switching pattern after radiotherapy exposure. <b>Results</b>: Gene-level analysis found 591 differentially expressed genes, with positive enrichment of PI3K/AKT-related pathways and negative enrichment of translation-associated processes. Isoform-level analysis uncovered 827 significant isoform switching events across 420 genes, often involving shifts from protein-coding transcripts to non-coding variants. Functional annotation showed that most isoform switches resulted in the loss of open reading frames and protein domains, especially in ribosomal proteins and translation machinery components. Exploratory proteomic analysis identified 191 differentially abundant proteins, including a significant reduction in RPL7A, providing partial protein-level support for translation-associated transcriptomic remodeling. <b>Conclusions</b>: RT-induced stress causes qualitative transcriptomic remodeling beyond changes in overall gene expression. These findings indicate that radiotherapy induces coordinated gene-, transcript-, and isoform-level changes in HMC3 cells. The alterations observed in translation-associated genes, together with partial proteomic concordance, identify candidate mechanisms for future functional investigation.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 4","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13516098/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148831227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Exploratory Study on Transcriptomic Profiling of Circulating miRNA Associated with Hub Genes in the Development of Polycystic Ovary Syndrome.","authors":"Yogesh Vetriselvan, Jayakumar Swetha, Manoranjani Murugan, Irisappan Ganesh, Vishnu Bhat Ballambattu, Pushpa Premanath Kotur, Deepa Shanmugam, Marcella Sherin Samuel, Sambandam Ravikumar","doi":"10.3390/ncrna12040030","DOIUrl":"10.3390/ncrna12040030","url":null,"abstract":"<p><p><b>Background:</b> Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that affects women of reproductive age, often associated with metabolic issues, causing infertility. Although it is common, the cause of PCOS remains unknown, and early diagnosis is challenging. MicroRNAs (miRNAs) serve as post-transcriptional regulators of gene expression, play a significant role in PCOS development, and have emerged as biomarkers for reproductive and metabolic disorders. However, research on miRNA signatures in the Indian population is limited. This pilot exploratory study aims to compare the candidate differentially expressed miRNAs (DE miRNAs) in serum samples from individuals with and without PCOS using high-throughput miRNA sequencing. <b>Methods:</b> In the study, patients with PCOS and age-matched controls were included; small RNAs were isolated from their serum, libraries were prepared, and the libraries were analyzed by next-generation sequencing. Bioinformatics analysis, including miRBase annotation, differential expression analysis, target prediction, GO/KEGG enrichment, and hub gene network analysis, was performed. <b>Results:</b> A total of 967 miRNAs were identified, with seven showing differential expression (log2FC > 1, <i>p</i> < 0.05). Among these, six miRNAs of hsa-miR-219a-2-3p, hsa-mir-384, hsa-miR-149-5p, hsa-miR-3182, hsa-miR-3960, and hsa-miR-4508 were upregulated, whereas hsa-mir-139 was downregulated. Functional enrichment and hub gene analyses identified key targets, including <i>TP53</i>, <i>FOXO1</i>, <i>HIF1A</i>, and <i>HDAC1</i>, that are crucial to the cell cycle, insulin signaling, and hypoxia. <b>Conclusions:</b> These preliminary findings identify candidate serum miRNA signatures in Indian women that may be associated with PCOS's reproductive and metabolic issues. These pilot study results suggest potential miRNAs and their target pathway links with PCOS that need validation in larger cohorts for diagnostic or therapeutic applications.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 4","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515866/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148831142","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fraction-Seq: An Integrated Experimental and Computational Workflow for Determining the Localization and Abundance of Small Non-Coding RNAs in Subcellular Compartments.","authors":"Siddhartha Shah, Tess Cherlin, Yi Jing, Stepan Nersisyan, Benjamin Leiby, Isidore Rigoutsos","doi":"10.3390/ncrna12040029","DOIUrl":"10.3390/ncrna12040029","url":null,"abstract":"<p><p>Small non-coding RNAs (sncRNAs) have garnered considerable attention in recent years, following accumulating evidence of their critical roles in many cellular processes. Among sncRNAs, microRNAs (miRNAs) and their isoforms (isomiRs), tRNA-derived fragments (tRFs), rRNA-derived fragments (rRFs), and Y RNA-derived fragments (yRFs) account for more than 95% of all sncRNAs found in cells. Despite their critical regulatory roles, most sncRNAs remain uncharacterized because their functionalization is a lengthy and challenging undertaking. Knowing an sncRNA's abundance helps prioritize among the various choices, while knowing where it localizes in the cell greatly limits the number and identity of its potential targets and helps understand its function. Most studies to date have assumed that the subcellular localization of the various sncRNA classes is understood and remains unchanged across cell types. However, as we have recently demonstrated, the subcellular distribution of sncRNAs follows complex patterns that depend on the sequence of the sncRNA, the presence or absence of post-transcriptionally added non-templated nucleotides, and the cell type. To determine the subcellular localization and abundance of sncRNAs and aid the design of targeted experimental studies of sncRNA function, we developed \"Fraction-seq.\" The method combines an experimental and an analytical component to remove cross-fraction contamination and reconstruct the true abundance of sncRNAs in each considered fraction. Fraction-seq can be applied to any adherent cells from any organism without modifications and can reconstruct the abundance of all categories of sncRNAs. Accompanying this detailed protocol is a newly developed port of the original SAS codes to the widely used R programming language. The codes and an example are freely available through our center's GitHub page.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 4","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13516553/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148831190","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Induced RNA Interference Can Suppress Persistent <i>Drosophila</i> A Virus (DAV) Infection in Cultured Drosophila Melanogaster S2 Cells.","authors":"Wiebke Kochendörfer, Klaus Förstemann","doi":"10.3390/ncrna12040028","DOIUrl":"10.3390/ncrna12040028","url":null,"abstract":"<p><p><b>Background</b>: The RNA interference (RNAi) pathway is a highly conserved antiviral mechanism in eukaryotes, including insects such as fruit flies. Many viruses have therefore evolved mechanisms that protect their transcripts and/or genomes; how effectively RNAi can act is thus a question that must be answered case-by-case. <b>Methods</b>: We demonstrate that the RNAi machinery can successfully combat <i>Drosophila</i> A virus (DAV) in persistently infected <i>Drosophila melanogaster</i> S2 cell lines, but only when supported with exogenous triggers. Since such an infection poses a challenge to the fitness of cells and the reproducibility of results obtained with them, our approach provides a convenient method to recover precious experimentally modified cell lines from infection through the application of exogenous double-stranded RNA (dsRNA) targeting the DAV sequence. The in vitro transcribed dsRNAs were applied to infected S2 cells via direct \"soaking,\" utilizing the cells' natural endocytic uptake. <b>Results</b>: We monitored treatment efficacy via RT-PCR-based detection of viral sequences, and our results demonstrate that continued application of DAV-specific dsRNAs led to a reduction in viral abundance and all eight tested cell lines appeared DAV-negative within several weeks of treatment. While a set of cell lines exhibited viral recurrence several weeks after the cessation of treatment, others remained virus-free for at least 16 weeks, suggesting that a permanent \"cure\" can indeed be achieved. Notably, while RNAi activation effectively cleared DAV in some cell lines, no beneficial effect was observed for a concomitantly applied <i>Drosophila</i> X-virus (DXV) treatment in the co-infected cultures. <b>Conclusions</b>: This suggests that DXV has more efficient evasion mechanisms but cannot protect DAV <i>in trans</i>. Our protocol thus provides a robust, scalable approach for clearing persistent DAV infections, but may not be effective against all viruses known to infect <i>Drosophila</i> cell cultures.</p>","PeriodicalId":19271,"journal":{"name":"Non-Coding RNA","volume":"12 4","pages":""},"PeriodicalIF":4.2,"publicationDate":"2026-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13516747/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148831240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}